Craniofacial defects are the second most common birth defect in the United States. A better understanding of the normal developmental processes that occur during craniofacial development is crucial. The neural crest is a vertebrate unique population of cells during development that gives rise to a diverse set of lineages, including craniofacial cartilage. Neural crest specification is controlled by a cascade of regulatory interactions between transcription factors, referred to as the neural crest specification gene regulatory network (GRN). Transcription factor activating protein 2 alpha (TFAP2A) is a key member of this GRN. Mutations in TFAP2A cause Branchio-occulo-facial syndrome (BOFS), which presents with cleft lip and or palate. The objective of my proposal is to dissect the regulatory interactions surrounding tfap2a in the neural crest specification GRN, using zebrafish as a model organism. First, I propose to dissect the cis-regulatory mechanisms used to control tfap2a expression in the neural crest. I hypothesize that pax, tcf, and tfap2 transcription factors act at an enhancer of tfap2a to activate its expression in the neural crest. To test this hypothesis, I propose the use of in vivo reporter assays to find enhancers of tfap2a. I will identify the necessary transcription factors by mutation of binding sites and in vivo reporter assays in loss of function backgrounds. I will use CRISPR/cas9 to delete the enhancer in zebrafish to determine its necessity for tfap2a expression in the neural crest. Second, I propose to identify the essential targets of tfap2a in the neural crest specification GRN. I hypothesize that sox10 and sox9b are the critical targets necessary for neural crest specification. To test this hypothesis, I propose loss of function and gain of function studies. I will analyze the craniofacial structures and gene expression changes in each of these paradigms. From these data, I will infer the relationships within this GRN. Finally, I propose to study the cis-regulatory mechanisms used for tfap2a to activate sox9b. These studies will reveal, in detail, connections in the neural crest specification GRN that are currently missing and further give a better understanding of the genetic underlying of the very early steps of craniofacial development.